Macrophages promote an early host response to infection by releasing pro-inflammatory cytokines such as interleukin (IL) 1β (IL-1β), tumour necrosis factor (TNF), and IL-6. One of the mechanisms through which cells sense pathogenic microorganisms is through Toll-like receptors (TLRs). IL-1 receptor-associated kinase (IRAK) 1, IRAK2, IRAK3, and IRAK4 are integral to TLR and IL-1 receptor signalling pathways. Recent studies suggest a role for aberrant TLR8 and NLRP3 inflammasome activation during both COVID-19 and HIV-1 infection. Here, we show that pacritinib inhibits the TLR8-dependent pro-inflammatory cytokine response elicited by GU-rich single-stranded RNA derived from SARS-CoV-2 and HIV-1. Using genetic and pharmacologic inhibition, we demonstrate that pacritinib inhibits IRAK1 phosphorylation and ubiquitination which then inhibits the recruitment of the TAK1 complex to IRAK1, thus inhibiting the activation of downstream signalling and the production of pro-inflammatory cytokines.

Macrophages are a reservoir for latent human immunodeficiency type 1 (HIV) infection and a barrier to HIV eradication. In contrast to CD4+ T cells, macrophages are resistant to the cytopathic effects of acute HIV infection. Emerging data suggest a role for TREM1 (triggering receptor expressed on myeloid cells 1) in this resistance to HIV-mediated cytopathogenesis. Here, we show that upon HIV infection, macrophages increase the expression of BCL2, BCLXL, TREM1, mitofusin 1 (MFN1), and MFN2 and the translocation of BCL2L11 (BIM) to the mitochondria and decrease the expression of BCL2-associated agonist of cell death (BAD) and BAX while maintaining a 95% survival rate over 28 days. The HIV proteins Tat and gp120 and the GU-rich single-stranded RNA (ssRNA) (RNA40) from the HIV long terminal repeat region (and a natural Toll-like receptor 8 [TLR8] agonist) induced similar effects. TREM1 silencing in HIV-infected macrophages led to decreased expression of BCL2, BCLXL, MFN1, and MFN2 and increased expression of BAD and BAX. This correlated with a significant increase in apoptosis mediated by a disruption of the mitochondrial membrane potential (Δψm), leading to the release of cytochrome c and caspase 9 cleavage. Exposure of TREM1-silenced macrophages to Tat, gp120, or RNA40 similarly resulted in the disruption of Δψm, cytochrome c release, caspase 9 cleavage, and apoptosis. Thus, our findings identify a mechanism whereby HIV promotes macrophage survival through TREM1-dependent upregulation of BCL2 family proteins and mitofusins that inhibits BCL2L11-mediated disruption of Δψm and subsequent apoptosis. These findings indicate that TREM1 can be a useful target for elimination of the HIV reservoir in macrophages.IMPORTANCE The major challenge to human immunodeficiency virus (HIV) treatment is the development of strategies that lead to viral eradication. A roadblock to accomplishing this goal is the lack of an approach that would safely eliminate HIV from all resting/latent reservoirs, including macrophages. Macrophages are a key part of the innate immune system and are responsible for recognizing invading microbes and sending appropriate signals to other immune cells. Here, we found that HIV induces the upregulation of the protein TREM1 (triggering receptor expressed on myeloid cells 1), which signals an increase in the expression of antiapoptotic proteins, thus promoting survival of HIV-infected macrophages.

Macrophages promote an early host response to infection by releasing pro-inflammatory cytokines such as interleukin-1β (IL-1β), TNF, and IL-6. The bioactivity of IL-1β is classically dependent on NLRP3 inflammasome activation, which culminates in caspase-1 activation and pyroptosis. Recent studies suggest a role for NLRP3 inflammasome activation in lung inflammation and fibrosis in both COVID-19 and SARS, and there is evidence of NLRP3 involvement in HIV-1 disease. Here, we show that GU-rich single-stranded RNA (GU-rich RNA) derived from SARS-CoV-2, SARS-CoV-1, and HIV-1 trigger a TLR8-dependent pro-inflammatory cytokine response from human macrophages in the absence of pyroptosis, with GU-rich RNA from the SARS-CoV-2 spike protein triggering the greatest inflammatory response. Using genetic and pharmacological inhibition, we show that the induction of mature IL-1β is through a non-classical pathway dependent on caspase-1, caspase-8, the NLRP3 inflammasome, potassium efflux, and autophagy while being independent of TRIF (TICAM1), vitamin D3, and pyroptosis.

HIV Nef acts as an anti-autophagic maturation factor through interaction with beclin-1 (BECN1). We report that exposure of macrophages to infectious or non-infectious purified HIV induces toll-like receptor 8 (TLR8) and BECN1 dependent dephosphorylation and nuclear translocation of TFEB and that this correlates with an increase in autophagy markers. RNA interference for ATG13, TFEB, TLR8, or BECN1 inhibits this HIV-induced autophagy. However, once HIV establishes a productive infection, TFEB phosphorylation and cytoplasmic sequestration are increased resulting in decreased autophagy markers. Moreover, by 7 d post-infection, autophagy levels are similar to mock infected controls. Conversely, although Nef deleted HIV similarly induces TFEB dephosphorylation and nuclear localization, and increases autophagy, these levels remain elevated during continued productive infection. Thus, the interaction between HIV and TLR8 serves as a signal for autophagy induction that is dependent upon the dephosphorylation and nuclear translocation of TFEB. During permissive infection, Nef binds BECN1 resulting in mammalian target of rapamycin (MTOR) activation, TFEB phosphorylation and cytosolic sequestration, and the inhibition of autophagy. To our knowledge, this is the first report of a virus modulating TFEB localization and helps to explain how HIV modulates autophagy to promote its own replication and cell survival.

Despite significant advances in the treatment of human immunodeficiency virus type-1 (HIV) infection, antiretroviral therapy only suppresses viral replication but is unable to eliminate infection. Thus, discontinuation of antiretrovirals results in viral reactivation and disease progression. A major reservoir of HIV latent infection resides in resting central memory CD4+ T cells (TCM) that escape clearance by current therapeutic regimens and will require novel strategies for elimination. Here, we evaluated the therapeutic potential of autophagy-inducing peptides, Tat-Beclin 1 and Tat-vFLIP-α2, which can induce a novel Na+/K+-ATPase dependent form of cell death (autosis), to kill latently HIV-infected TCM while preventing virologic rebound. In this study, we encapsulated autophagy inducing peptides into biodegradable lipid-coated hybrid PLGA (poly lactic-co-glycolic acid) nanoparticles for controlled intracellular delivery. A single dose of nanopeptides was found to eliminate latent HIV infection in an in vitro primary model of HIV latency and ex vivo using resting CD4+ T cells obtained from peripheral blood mononuclear cells of HIV-infected patients on antiretroviral with fully suppressed virus for greater than 12 months. Notably, increased LC3B lipidation, SQSTM1/p62 degradation and Na+/K+-ATPase activity characteristic of autosis, were detected in nanopeptide treated latently HIV-infected cells compared to untreated uninfected or infected cells. Nanopeptide-induced cell death could be reversed by knockdown of autophagy proteins, ATG5 and ATG7, and inhibition or knockdown of Na+/K+-ATPase. Importantly, viral rebound was not detected following the induction of the Na+/K+-ATPase dependent form of cell death induced by the Tat-Beclin 1 and Tat-vFLIP-α2 nanopeptides. These findings provide a novel strategy to eradicate HIV latently infected resting memory CD4+ T cells, the major reservoir of HIV latency, through the induction of Na+/K+-ATPase dependent autophagy, while preventing reactivation of virus and new infection of uninfected bystander cells.

Because microglia are a reservoir for HIV and are resistant to the cytopathic effects of HIV infection, they are a roadblock for any HIV cure strategy. We have previously identified that triggering receptor expressed on myeloid cells 1 (TREM1) plays a key role in human macrophage resistance to HIV-mediated cytopathogenesis. In this article, we show that HIV-infected human microglia express increased levels of TREM1 and are resistant to HIV-induced apoptosis. Moreover, upon genetic inhibition of TREM1, HIV-infected microglia undergo cell death in the absence of increased viral or proinflammatory cytokine expression or the targeting of uninfected cells. We also show that the expression of TREM1 is mediated by HIV Tat through a TLR4, TICAM1, PG-endoperoxide synthase 2, PGE synthase, and PGE2-dependent manner. These findings highlight the potential of TREM1 as a therapeutic target to eradicate HIV-infected microglia without inducing a proinflammatory response.

Human immunodeficiency type 1 (HIV)-infected macrophages (HIV-Mφ) are a reservoir for latent HIV infection and a barrier to HIV eradication. In contrast to CD4+ T cells, HIV-Mφ are resistant to the cytopathic effects of acute HIV infection and have increased expression of cell survival factors, including X-linked inhibitor of apoptosis (XIAP), baculoviral IAP repeat containing (BIRC) 2/cIAP1, beclin-1, BCL2, BCL-xl, triggering receptor expressed on myeloid cells 1, mitofusin (MFN) 1, and MFN2. DIABLO/SMAC mimetics are therapeutic agents that affect cancer cell survival and induce cell death. We found that DIABLO/SMAC mimetics (LCL-161, AT-406 (also known as SM-406 or Debio 1143), and birinapant) selectively kill HIV-Mφ without increasing bystander cell death. DIABLO/SMAC mimetic treatment of HIV-Mφ-induced XIAP and BIRC2 degradation, leading to the induction of autophagy and the formation of a death-inducing signaling complex on phagophore membranes that includes both pro-apoptotic or necroptotic (FADD, receptor-interacting protein kinase (RIPK) 1, RIPK3, caspase 8, and MLKL) and autophagy (ATG5, ATG7, and SQSTM1) proteins. Genetic or pharmacologic inhibition of early stages of autophagy, but not late stages of autophagy, ablated this interaction and inhibited apoptosis. Furthermore, DIABLO/SMAC mimetic-mediated apoptosis of HIV-Mφ is dependent upon tumor necrosis factor signaling. Our findings thus demonstrate that DIABLO/SMAC mimetics selectively induce autophagy-dependent apoptosis in HIV-Mφ.

Long-lived resting memory CD4+ T cells (TCM) are a major reservoir of latent HIV infection. We hypothesized that latent HIV-TCM cells are maintained by aberrant expression of cell survival factors, including XIAP, BIRC2/cIAP1, and beclin-1. DIABLO/SMAC mimetics are therapeutic agents that compromise cell survival by hijacking host apoptotic machinery. We found that DIABLO/SMAC mimetics (birinapant, GDC-0152, and embelin) selectively kill HIV-TCM without increasing virus production or targeting uninfected TCM. Treatment of HIV-TCM with DIABLO/SMAC mimetics promoted XIAP and BIRC2 degradation, which triggered autophagy and the formation of a cell death complex consisting of pro-apoptotic (FADD, RIPK1, RIPK3, and caspase 8) and autophagy (ATG5, ATG7, and SQSTM1) proteins. Genetic or pharmacological inhibition of autophagy induction, but not autophagy-mediated degradation, abrogated this interaction and subsequent cell death. Our findings identify a mechanism whereby DIABLO/SMAC mimetics exploit autophagy and apoptotic machinery to selectively induce killing of HIV-TCM without viral reactivation while sparing uninfected cells.

HIV infection and progression to AIDS is characterized by the depletion of T cells, which could be due, in part, to apoptosis mediated by the extra-cellular HIV-encoded Tat protein as a consequence of Tat binding to tubulin. Microtubules are tubulin polymers that are essential for cell structure and division. Molecules that target microtubules induce apoptosis and are potent anti-cancer drugs. We studied the effect on tubulin polymerization of three Tat variants: Tat HxB2 and Tat Eli from patients who are rapid progressors (RP) and Tat Oyi from highly exposed but persistently seronegative (HEPS) patients. We compared the effect on tubulin polymerization of these Tat variants and peptides corresponding to different parts of the Tat sequence, with paclitaxel, an anti-cancer drug that targets microtubules.

The HIV-1 Tat protein is a promising target to develop AIDS therapies, particularly vaccines, due to its extracellular role that protects HIV-1-infected cells from the immune system. Tat exists in two different lengths, 86 or 87 residues and 99 or 101 residues, with the long form being predominant in clinical isolates. We report here a structural study of the 99 residue Tat Eli variant using 2D liquid-state NMR, molecular modeling and circular dichroism.

Therapeutic strategies that provide effective and broad-spectrum neutralization against HIV-1 infection are highly desirable. Here, we investigate the potential of nanoengineered CD4+ T cell membrane-coated nanoparticles (TNP) to neutralize a broad range of HIV-1 strains. TNP displayed outstanding neutralizing breadth and potency; they neutralized all 125 HIV-1-pseudotyped viruses tested, including global subtypes/recombinant forms, and transmitted/founder viruses, with a geometric mean 80% inhibitory concentration (IC80) of 819 μg ml-1 (range, 72 to 8,570 μg ml-1). TNP also selectively bound to and induced autophagy in HIV-1-infected CD4+ T cells and macrophages, while having no effect on uninfected cells. This TNP-mediated autophagy inhibited viral release and reduced cell-associated HIV-1 in a dose- and phospholipase D1-dependent manner. Genetic or pharmacological inhibition of autophagy ablated this effect. Thus, we can use TNP as therapeutic agents to neutralize cell-free HIV-1 and to target HIV-1 gp120-expressing cells to decrease the HIV-1 reservoir.IMPORTANCE HIV-1 is a major global health challenge. The development of an effective vaccine and/or a therapeutic cure is a top priority. The creation of vaccines that focus an antibody response toward a particular epitope of a protein has shown promise, but the genetic diversity of HIV-1 hinders this progress. Here we developed an approach using nanoengineered CD4+ T cell membrane-coated nanoparticles (TNP). Not only do TNP effectively neutralize all strains of HIV-1, but they also selectively bind to infected cells and decrease the release of HIV-1 particles through an autophagy-dependent mechanism with no drug-induced off-target or cytotoxic effects on bystander cells.

HIV-1 is a major global health challenge. The development of an effective vaccine and a therapeutic cure are top priorities. The creation of vaccines that focus an antibody response toward a particular epitope of a protein has shown promise, but the genetic diversity of HIV-1 stymies this progress. Therapeutic strategies that provide effective and broad-spectrum neutralization against HIV-1 infection are highly desirable. Methods: We investigated the potential of nanoengineered CD4+ T cell membrane-coated nanoparticles (TNP) encapsulating the DIABLO/SMAC mimetics LCL-161 or AT-406 (also known as SM-406 or Debio 1143) to both neutralize HIV-1 and selectively kill HIV-1-infected resting CD4+ T cells and macrophages. Results: DIABLO/SMAC mimetic-loaded TNP displayed outstanding neutralizing breadth and potency, and selectively kill HIV-1-infected cells via autophagy-dependent apoptosis while having no drug-induced off-target or cytotoxic effects on bystander cells. Genetic inhibition of early stages of autophagy abolishes this effect. Conclusion: DIABLO/SMAC mimetic loaded TNP have the potential to be used as therapeutic agents to neutralize cell-free HIV-1 and to kill specifically HIV-1-infected cells as part of an HIV-1 cure strategy.

The inhibitor of apoptosis protein BIRC2 regulates fundamental cell death and survival signaling pathways. Here we show that BIRC2 accumulates in the nucleus via binding of its second and third BIR domains, BIRC2BIR2 and BIRC2BIR3, to the histone H3 tail and report the structure of the BIRC2BIR3-H3 complex. RNA-seq analysis reveals that the genes involved in interferon and defense response signaling and cell-cycle regulation are most affected by depletion of BIRC2. Overexpression of BIRC2 delays DNA damage repair and recovery of the cell-cycle progression. We describe the structural mechanism for targeting of BIRC2BIR3 by a potent but biochemically uncharacterized small molecule inhibitor LCL161 and demonstrate that LCL161 disrupts the association of endogenous BIRC2 with H3 and stimulates cell death in cancer cells. We further show that LCL161 mediates degradation of BIRC2 in human immunodeficiency virus type 1-infected human CD4+ T cells. Our findings provide mechanistic insights into the nuclear accumulation of and blocking BIRC2.

Human immunodeficiency virus (HIV)-associated neurocognitive disorders (HAND) are a common source of morbidity in people living with HIV (PLWH). Although antiretroviral therapy (ART) has lessened the severity of neurocognitive disorders, cognitive impairment still occurs in PLWH receiving ART. The pathogenesis of HAND is likely multifaceted, but common factors include the persistence of HIV transcription within the central nervous system, higher levels of pro-inflammatory cytokines in the cerebrospinal fluid, and the presence of activated microglia. Toll-like receptor (TLR) 7 and TLR8 are innate pathogen recognition receptors located in microglia and other immune and non-immune cells that can recognise HIV RNA and trigger pro-inflammatory responses. IL-1 receptor-associated kinase (IRAK) 1 is key to these signalling pathways. Here, we show that IRAK1 inhibition inhibits the TLR7 and TLR8-dependent pro-inflammatory response to HIV RNA. Using genetic and pharmacological inhibition, we demonstrate that inhibition of IRAK1 prevents IRAK1 phosphorylation and ubiquitination, and the subsequent recruitment of TRAF6 and the TAK1 complex to IRAK1, resulting in the inhibition of downstream signalling and the suppression of pro-inflammatory cytokine and chemokine release.